Genetic diversity and population structure among native, naturalized, and invasive populations of the common yellow monkeyflower, Mimulus guttatus (Phrymaceae)

Abstract An ongoing controversy in invasion biology is the prevalence of colonizing plant populations that are able to establish and spread, while maintaining limited amounts of genetic variation. Invasive populations can be established through several routes including from a single source or from multiple introductions. The aim of this study was to examine genetic diversity in populations of Mimulus guttatus in the United Kingdom, where the species is considered invasive, and compare this diversity to that in native populations on the west coast of North America. Additionally, we looked at diversity in non‐native populations that have not yet become invasive (naturalized populations) in eastern North America. We investigated population structure among populations in these three regions and attempted to uncover the sources for populations that have established in the naturalized and invasive regions. We found that genetic diversity was, on average, relatively high in populations from the invasive UK region and comparable to native populations. Contrastingly, two naturalized M. guttatus populations were low in both genetic and genotypic diversity, indicating a history of asexual reproduction and self‐fertilization. A third naturalized population was found to be a polyploid Mimulus hybrid of unknown origin. Our results demonstrate that M. guttatus has likely achieved colonization success outside of its native western North America distribution by a variety of establishment pathways, including those with genetic and demographic benefits resulting from multiple introductions in the UK, reproductive assurance through selfing, and asexual reproduction in eastern North America, and possible polyploidization in one Canadian population.


| INTRODUC TI ON
Establishment by non-native plant species around the globe is a well-documented occurrence, and the probability of successfully colonizing a novel location outside of the species' native range depends on many variables. Factors that can restrict the establishment and spread of introduced plant populations include low genetic diversity and Allee effects associated with founder events, insufficient propagule pressure in the form of a single or few introductions, maladaptation to novel environmental pressures, or some combination of these (Lee, 2002;Lockwood et al., 2013;Richardson et al., 2000;Szczecinska et al., 2016;Williamson & Fitter, 1996). However, when a nascent plant population is able to overcome the influence of a novel suite of environmental pressures, the formation of establishment pathways can lead to a stage in the invasion process called naturalization.
According to Richardson et al. (2000), naturalized populations are those non-native plant populations that maintain sufficient population size by sexual reproduction or asexual vegetative proliferation, so that the probability of extinction due to environmental stochasticity is low. Naturalization is often considered an intermediate stage prior to a population becoming invasive, representing a lag phase of slow population growth as it deals with deficiencies inherent to a novel population's demographics or to maladaptation (Aikio et al., 2010;Frappier et al., 2004;Murren et al., 2009;Richardson & Pyšek, 2012). The naturalization stage is considered in many theoretical models of invasion a critical point in determining whether a non-native population goes extinct, remains cryptic and benign, or alternatively adapts and spreads aggressively into new locations (Catford et al., 2009;Guo et al., 2018;Richardson & Rejmánek, 2011). Despite the importance of the naturalization stage in characterizing the progression from casual colonization to impactful invasion in predictive models, few empirical studies include genetic diversity data from naturalized populations to compare with native and invasive populations (Pysek et al., 2008). This gap in our understanding is largely due to the difficulty in locating and recognizing naturalized populations prior to their becoming invasive (Aikio et al., 2010).
The important transition from naturalization to invasion is often dictated by the genetic constitution of the plant population, which is in turn governed by the mode of reproduction and mating system of the plant species in question (Garcia-Ramos & Rodriguez, 2002;Kinlan & Hastings, 2005). Invasive plant species display extensive variation regarding the importance of sexual versus asexual reproduction, and the degree to which sexual reproduction relies on outcrossing (i.e., mating between unrelated individuals) versus self-fertilization (Barrett et al., 2008). The mode of reproduction determines establishment and invasion potential because it influences population genetic parameters such as the amount of additive genetic variation, effective population size, and partitioning of genetic diversity within and among populations (Barrett, 1998;Eckert et al., 2010;Lopez-Villalobos & Eckert, 2018;Vogler & Kalisz, 2001).
Invasion processes have resulted in transitions to higher rates of self-fertilization in mixed-mating plants (e.g., selfing and asexual reproduction) in the introduced range (Barrett, 2011;Barrett et al., 2008;Clements & Ditommaso, 2011). The study of selffertilization and clonality and their role in facilitating colonization of non-native locations goes back decades to "Baker's Law" (Baker, 1955;Stebbins, 1957). Baker suggested that self-compatible species should theoretically be more successful colonizers following long-distance dispersal compared with obligate outcrossing species, in part because the former would need only one individual to establish a naturalized population (Kolar & Lodge, 2001). Asexual reproduction can also contribute to an introduced plant population's establishment and persistence in heterogenic environments, such as riparian ecosystems or roadside seeps (Cushman & Gaffney, 2010) that often results in a single or few genotypes expanding in an area, evidenced by the most successful aquatic plant invader, the water hyacinth, Eichhornia crassipes (Zhang et al., 2010).
While a single introduction, followed by some form of uniparental reproduction, has been shown to be a successful strategy to become naturalized, a more common scenario appears to be by multiple introductions of plant propagules followed by at least occasional outcrossing (Dlugosch & Parker, 2008;Wilson et al., 2009).
When outcrossing is the primary mode of reproduction, additive genetic variation within the population increases compared with populations that rely on selfing that results in a 50% decrease in heterozygosity after each generation (Carr & Dudash, 2003;Charlesworth & Charlesworth, 1987). By enhancing genetic variation, outcrossing enables an incipient population to respond and adapt more quickly to the changes in environmental conditions common during invasion (Barrett et al., 2008;Charlesworth, 2003;Lynch & Walsh, 1998). Outcrossing can also result in interspecific hybridization among closely related species that have recently come into contact, and examples of allopolyploid species such as Tragopogon mirus (Soltis et al., 2004), Senecio cambrensis (Abbott & Lowe, 2004), and a Spartina hybrid cross between the S. foliosa and S. alterniflora (Ainouche et al., 2009;Ayres et al., 2004) have been described.
In this study, we use molecular data to examine the genetic diversity and structure among a range of native and non-native populations of diverse origins and residence times. Specifically, we compare two naturalized eastern North American populations of the mixed-mating plant species, Mimulus guttatus D.C.
(Phrymaceae), a nearby naturalized population comprised of a heretofore-undescribed hybrid Mimulus taxon, three non-native M.
guttatus populations in the United Kingdom, where the species is considered invasive, and native populations that occur across a large span of the species' home range in western North America. Our goal was to address the following questions: (1) How does genetic and genotypic diversity in non-native populations (i.e., naturalized and invasive populations) compare with diversity in native populations?
We predicted that populations in the invasive region will have similar levels of genetic diversity as native populations due to the species' history of multiple introductions as an ornamental plant (Truscott et al., 2008); (2) Which native location is most likely the source for non-native M. guttatus populations? We predicted that non-native M. guttatus populations in the UK are derived from populations on the northern edge of the native distribution based on prior evidence (Puzey & Vallejo-Marín, 2014). There has been no prior investigation regarding the source region for naturalized populations on the east coast of North America, and we aim to shed light on the origin of these non-native populations.

| Study species
Mimulus guttatus (2n = 2x = 28), or common monkeyflower, is a herbaceous species native to the west coast of North America, found from Mexico to Alaska Dudash et al., 1997;Kelly & Arathi, 2003;Lowry et al., 2008;Wu et al., 2008). In the United Kingdom (UK), M. guttatus is considered a harmful invasive that was intentionally introduced as a horticultural species approximately 200 years ago (Truscott et al., 2008;van Kleunen & Fischer, 2008).

Recently, naturalized populations in New York State and New
Brunswick, Canada, have received attention (Murren et al., 2009).
Little is known of the evolutionary history of these naturalized populations, but they are thought to have established at least 50 years ago. Native populations of M. guttatus are described as mixedmating and exhibit wide variation in outcrossing rates (Dudash & Carr, 1998;Ritland & Ganders, 1987). To our knowledge, outcrossing rates in naturalized and invasive populations have not been estimated. Mimulus guttatus is also capable of asexual reproduction via fragmentation and stolons (Grant, 1924;Truscott et al., 2006;Vickery, 1959). Mimulus guttatus has become a model system in studies of ecological and evolutionary genomics because of its broad phenotypic and genetic diversity (Dudash et al., 2005;Wu et al., 2008). In its native range, M. guttatus populations can be found as either annuals or perennials, and this difference in life history depends on water availability (van Kleunen, 2007;Lowry et al., 2008; M. R. Dudash & C. J. Murren, unpublished data). For this study, we only sampled from perennial native populations because all known non-native populations are perennial.

| Sampling sites
To compare genetic diversity and population structure among M. guttatus populations in the native, naturalized, and invasive ranges, we conducted fieldwork in 2012 and 2013 (Table 1 includes  local contacts. In the naturalized region on the east coast of North America, two of the three populations sampled were previously studied by Murren et al. (2009) and located in Fly Creek, New York and Springfield, New Brunswick, Canada. Local botanists provided the location of a third population, near Bass River, New Brunswick (NBBR). Initially, the NBBR population was thought to be comprised of M. guttatus individuals. However, following assessment of genotyping data, chromosome counts, and morphological traits (e.g., low pollen viability and reduced seed set in the greenhouse), it was apparent that the NBBR population was comprised of polyploid hybrid Mimulus individuals. These individuals likely represent a polyploid (2n = 3x = 42-46; J. A. Berg, unpublished data) with M. guttatus constituting at least one of the parental taxa. It has been shown that M. guttatus readily hybridizes with closely related Mimulus species to form allopolyploids (Clausen et al., 1950;Vallejo-Marin, 2012;Vickery, 1978), including the triploid hybrid M. × robertsii Silverside (2n = 3x = 44-46) formed by M. guttatus and a South American species, M. luteus, which is also found throughout the UK (Vallejo-Marin & Lye, 2013). The second parent taxon of the NBBR hybrid population is likely a tetraploid Mimulus species, but its identification was beyond the scope of this study. Here, we utilize the NBBR population in the surveys of genetic diversity, but do not include it in the structure analyses due to the difficulties that arise in combining polyploid and diploid data in these assessments.
For each of the 14 native and naturalized populations, we randomly sampled fruits and leaf tissue from 30 to 50 individual plants that were >1 m apart to increase the chances of sampling multiple genotypes. Leaf tissue was immediately stored in silica gel until DNA extraction. For the three M. guttatus populations in the invasive region in the UK, we obtained field-collected seed from a colleague, Mario Vallejo-Marin, at the University of Stirling in Scotland. This seed was then grown at the University of Maryland (UMD) greenhouse, and when seedlings were ~6 cm tall, leaf tissue was collected from 20 individuals per population and stored in silica gel. Following DNA extraction and genotyping (see below), the sample size was reduced to 14 individuals for each of the UK populations due to poorquality DNA in some samples.

| Genetic markers
To genotype the 17 populations from the native, naturalized, and invasive regions, we used 12 codominant markers (Table 2)

| DNA extraction PCR amplification
To extract DNA from leaf tissue, a modified CTAB protocol (Doyle & Doyle, 1990) was employed on an AutoGenprep 965/960 instrument (AutoGen) using the Plant DNA Extraction Kit AGP965/960, following the manufacturer's protocol. DNAs were amplified for the 12 loci in sets of two multiplexed reactions (  (Brody & Kern, 2004). PCR products were diluted in nuclease-free water (dilutions ranged from 1:10 to 1:50), and 1 μl of each dilution was added to 9 μl of HiDi formamide with 1 μl ROX standard (DeWoody et al., 2004). Samples were heated to 95°C for 6 min, cooled to 4°C for 6 min, and loaded onto an ABI 3730xl automated capillary sequencer with a 50 cm, 96 channel array containing POP-7 polymer for fragment analysis at the

Laboratories of Analytical Biology (LAB) of the Smithsonian National
Museum of Natural History.

| Genetic analyses
We performed allele binning and analyzed raw peak sizes from fluorescent fragment profiles using GeneMapper v5.0 software (Applied Biosystems), which allows calling of multiple peaks per locus. A random sample of 10% of individuals was reassayed and rescored to check consistency. For the polyploid individuals at the NBBR site, determining conventional genetic diversity parameters based on allele frequency (e.g., expected heterozygosity) is problematic because of the difficulty in identifying alleles in partial heterozygotes. Therefore, to assess allelic diversity in each population and between regions, we calculated the following statistics ( the number of private alleles (P). Welch's two-sample t-tests were used to test for significant differences between regions for these statistics (the NBBR population containing polyploid individuals was excluded from t-tests). For the diploid M. guttatus populations, we used GenAlEx 6.5 to calculate expected heterozygosity and deviations from Hardy-Weinberg equilibrium (HWE). To determine pairwise genetic differences between individuals within each population, we used the method developed for microsatellite data by Bruvo et al. (2004) in the R package poppr (Kamvar et al., 2014). The distance measure of Bruvo et al. (2004) is similar to band-sharing indices and is appropriate for relative distance comparison among intraspecific individuals of different ploidy levels and takes into account stepwise mutational processes.
We used poppr for multilocus genotype (MLG) assignment, to determine expected proportion of MLGs from the total number of individuals sampled (R) using a rarefaction method to account for sample size (Hurlbert, 1971) and to calculate the complement of Simpson's diversity index D (Simpson, 1949).

| Population genetic structure
We The second analysis, DAPC, is a multivariate method to identify clusters comprised of genetically similar individuals (Jombart et al., 2010). DAPC uses principle components derived from principle components analysis (PCA) as variables to optimize betweengroup variation and minimize within-group variation in order to separate individuals into predefined groups (Jombart et al., 2010).
The method uses a k-means clustering algorithm to analyze any number of potential clusters (k's) in a sequential manner. The optimal k should correspond with the lowest Bayesian Information Criterion (BIC) score. DAPC has been suggested as an alternative to other Bayesian clustering methods such as STRUCTURE (Pritchard et al., 2000) because it does not require a population genetic model to identify clusters. Therefore, DAPCs are suitable for analyzing complex genetic data sets such as those that may not adhere to the assumption of random mating within populations (e.g., clonality or self-fertilization). The DAPC analysis was conducted in the R package adegenet 1.3-1 (Jombart & Ahmed, 2011).
Prior to DAPC analysis, we used the adegenet function clonecorrect to account for clonality in the data set. Next, to find the optimal TA B L E 3 Measures of genotypic and genetic diversity of 16 Mimulus guttatus populations and 1 polyploid Mimulus hybrid population (NBBR) sampled from three regions, native (western North America), naturalized (eastern North America), and invasive (United Kingdom).  number of clusters, we used k-means clustering of principal components using the function find. clusters in adegenet. The function xvalDapc was used to cross-validate the number of principal components used in the analysis.
To complement the DAPC analysis and help resolve structure among the 16 M. guttatus populations, we constructed a dendrogram based on Nei's genetic distance (Nei, 1978). Data were bootstrapped in the R package poppr using the aboot function from a sample of 1000 bootstrapped trees. The function clonecorrect was applied to the data prior to bootstrapping to account for clonality within populations.    Table 3).
The mean pairwise genetic difference between individuals (H′;   Table 3) indices (0.94 ± 0.01 and 0.93 ± 0, respectively), and both indices were more than twice that found in the naturalized region (0.46 ± 0.03) and nearly threefold larger than the D calculated for NBBR (0.34).

| Population genetic structure
An AMOVA (

The discriminant analysis of principal components (DAPC)
showed that the k-means clustering separated the data set (  To examine the relationship between genetic and geographic distance, we performed three Mantel tests (r m ; Table 5

| DISCUSS ION
Understanding the role of genetic variation in plant invasions is a primary focus in modern ecology, and reconciling the paradox that exists when plant populations with low genetic diversity are able to establish in non-native locations is a goal for researchers interested in managing these populations (Dlugosch & Parker, 2008;Lee, 2002;Moran & Alexander, 2014).

| Genetic and genotypic diversity in native, naturalized, and invasive regions
Our comparison of genetic diversity revealed that in the two naturalized M. guttatus populations, NBS and FC, diversity was substantially lower, on average, than that found in populations located in the native region. While these differences were not statistically significant at the p = .05 level (due in part to the low level of detectable genetic variability in the naturalized region), we found that the average total number of alleles, number of alleles per locus, and observed heterozygosity in native populations were nearly twice that found in the two naturalized populations (Table 3) Note: A significant correlation (r m ) indicates isolation by distance. An asterisk indicates a significant results at the p < .01 level.
non-native locations. Perhaps the two MLGs in the NBS population that we uncovered in this study were selected for certain adaptive plastic traits that suited them well following introduction into the remote location in Springfield, New Brunswick, Canada. Given the low genetic variation and genotypic diversity in the NBS population, a logical next step would be to examine these individuals for their capacity to express plasticity in novel, stressful environments. A companion greenhouse study was conducted to shed light on the role of phenotypic plasticity in response to abiotic conditions that naturally vary among native and non-native populations across their distribution in this study.
The guttatus from rapid population growth.
The second naturalized M. guttatus population in this study, FC (Fly Creek, New York), was also relatively deficient in some measures of genetic diversity relative to native populations but overall its observed heterozygosity was similar to the average heterozygosity found in the native populations. The FC population may have a similar introduction history as the NBS population, but it is unlikely they originated from the same source population (see discussion of source populations below). Methods designed to detect a recent reduction in population size based on the principle of excess heterozygosity using microsatellites or other molecular markers (Beaumont, 1999;Cornuet & Luikart, 1996;Garza & Williamson, 2001) typically require larger sample sizes than the six MLGs representing FC in this study in order to obtain robust statistical results. Therefore, based on our findings, further sampling is warranted to determine whether the naturalized M. guttatus population located in Fly Creek, New York, may be the product of a recent bottleneck, especially given our estimate of its population size of greater than 1000 individuals.
In the three populations from the invasive region in the UK, genetic and genotypic diversity was similar to the native populations, supporting the evidence of multiple introductions in this region and suggesting that outcrossing is the prominent mode of reproduction.
Mimulus guttatus was introduced into the UK repeatedly as a horticultural species (Truscott et al., 2008;van Kleunen & Fischer, 2008), pressure has been found to be a common denominator explaining nearly all successful invasions for which there are historical records of introduction (Colautti et al., 2006). Based on the scope of our study, we cannot say definitively that the greater genotypic diversity found in M. guttatus populations from the invasive region compared with the naturalized region provides an ample explanation as to why some M. guttatus populations become invasive while others do not.
However, our results can be used in combination with future environmental comparisons and evaluations of residence times (Pysek et al., 2009)  We found no strong association that would identify any of the 11 native M. guttatus populations as a source for any non-native populations in our study, although there was slight evidence for the native Alaskan group being the most closely related to the group comprised of the three invasive UK populations and the NBS population (Figure 3). This would support a prior study that identified the northern edge of the native distribution as the potential source for introduced populations in the UK. Using genome resequencing data, Puzey and Vallejo-Marín (2014) (Cristescu, 2015).
As mentioned above, we found some support for the inclusion of the naturalized NBS population within the clade formed by the UK populations in the invasive region (Figure 3,  Thus, if certain genotypes are more successful colonizers, either because they express traits that allow for persistence during founder effects (e.g., low inbreeding depression and exploitation of resources following disturbance) or they fit Baker's description of the "general-purpose genotype" by being more phenotypically plastic than other genotypes (Baker, 1974), then the bridgehead effect could act as an efficient process for choosing adaptive colonizers that can leapfrog into other territories. It is plausible that the source population for the NBS population in New Brunswick, Canada, is located in Europe, as the two continents maintain a robust trade in horticultural products. From 2013 to 2015, the EU exported over $9 billion in horticultural products to the US alone.
It is possible that the founding propagules that colonized the NBS site arrived from Europe prior to the enforcement of current efforts such as the USDA Plant Protection and Quarantine program, enacted to restrict the import of potentially invasive plant species.  On the east coast of North America, neither M. cupreus or M. luteus has been recorded. The other yellow-flowered species that has been found is M. moschatus, a non-native escape from garden plots (Pennell, 1935). Mimulus moschatus is a tetraploid (2n = 4x = 32), which makes it a candidate as the second parental taxon with M.
guttatus that could produce the putative triploid population NBBR.
However, the results from chromosome counts revealed higher counts than would be expected from an M. guttatus × M. moschatus hybrid, and they have also been reported as being incompatible (Vallejo-Marin, 2012).
Because most triploid Mimulus hybrids in the UK have been found to be largely sterile (Vallejo-Marin, 2012;Vallejo-Marin & Lye, 2013), the NBBR population may represent a pathway to establishment in the naturalized region directed by uniparental asexual reproduction. Its sterility and the fact that few potential parent Mimulus species occur on the east coast of North America to propagate more hybrids means that additional colonization by this hybrid would have to come from emigrants from the present population or future escapees. More research is required to definitively identify this population/species and its ploidy level before we can make accurate assessments concerning its potential to progress from a naturalized population to one that may begin to spread and become invasive on the east coast of North America.

| CON CLUS ION
Our study of M. guttatus populations from native, naturalized, and invasive regions demonstrates that naturalized populations in eastern North America have low genetic and genotypic variation compared with native populations on the west coast of North America. It is likely that these two naturalized populations experienced founder effects and rely on uniparental reproduction, asexual reproduction, and/or selfing, to persist. A third naturalized population in New Brunswick, Canada, was identified as a polyploid Mimulus species and may demonstrate interspecific hybridization as a successful pathway to establishment in remote novel areas. Populations in the invasive region in the UK have similar genetic and genotypic diversity as the native populations, an expected result because of their historical record of multiple introductions. The invasive region may have also served as the source population for the naturalized population, NBS, providing a possible example of the bridgehead effect.
More work is required to determine whether naturalized populations are restricted from becoming invasive because they lack genetic variation, or because they are limited by environmental factors. By continuing to monitor these naturalized populations, we can learn much about the invasion process while controlling their potential spread.

ACK N OWLED G M ENTS
We

DATA AVA I L A B I L I T Y S TAT E M E N T
Microsatellite profile information is available at https://doi. org/10.5061/dryad.cvdnc jt7c.